Beneficial effects of flavonoids on animal models of atherosclerosis: A systematic review and meta-analysis

Summary Atherosclerosis is the main cause of cardiovascular diseases that seriously endanger human health. The existing treatment drugs are effective, but they have some side effects. Accumulating evidence suggests that flavonoids have attracted wide attention due to their multiple cardioprotective effects and fewer side effects. PubMed, Web of Science database, Embase, and Cochrane Library were searched for studies evaluating the effects of flavonoids against atherosclerosis. 119 studies published from August 1954 to April 2023 were included. Random-effects models were performed for synthesis. Compared with the control group, flavonoids significantly reduced longitudinal and cross-sectional plaque area. The findings indicated that flavonoids significantly reduced the concentrations of serum TC, TG, and LDL-C and increased serum HDL-C concentrations. Besides, flavonoids reduced the levels of circulating pro-inflammatory factors, including TNF-α, IL-1β, and IL-6, and increased the serum IL-10 level. This study provides evidence for the potential cardiovascular benefits of flavonoids.


Highlights
It is aimed to evaluate the effects of flavonoids against atherosclerosis A systematic review and meta-analysis of 119 studies are conducted It was found that flavonoids exert anti-atherosclerotic effects in animal models

INTRODUCTION
Atherosclerosis and subsequent coronary heart diseases (CHD) are major health concerns and the leading causes of morbidity and mortality worldwide. 1Anti-atherosclerosis studies to prevent the development of CHD have become a research hotspot.Atherosclerosis is a chronic inflammatory disease characterized by dysregulation of lipid metabolism and the formation of atherosclerotic plaques in the vessel wall. 2,3therosclerosis's progression is drastically expedited by high levels of low-density lipoprotein cholesterol (LDL-C) and plaque rupture-induced thrombosis. 4In view of this, the primary strategies for preventing and treating atherosclerosis include lipid-lowering, anti-inflammation, antithrombosis, and re-establishment of arterial flow. 4,5The existing treatment drugs, including lipid-regulating drugs, anti-inflammation drugs, antiplatelet drugs, and thrombolytic anticoagulant drugs, are effective but inevitably have side effects such as bleeding and liver and kidney damage.Therefore, it is of great clinical significance to actively explore safe and effective therapeutic drugs for atherosclerosis.
In recent years, traditional Chinese medicine has been found to have multiple cardioprotective effects 6 and has been widely used in the treatment of atherosclerosis in China and many other Asian countries. 7A growing body of studies has shown that flavonoids, as a kind of traditional Chinese medicine monomer, have an anti-atherosclerotic effect with a few side effects. 80][11] Based upon structural differences, flavonoids can be generally classified into seven major subclasses: flavonols, flavones, flavanols (flavan-3-ols or catechins), flavanones, anthocyanidins, isoflavones, and chalcones. 12,13Studies have reported that flavonoids have a variety of biological activities, including anti-cancer, anti-inflammatory, cardiac protection, neuroprotection, etc. [13][14][15] The result of a multi-center randomized controlled trial suggested that flavonoids might exert anti-atherosclerosis effects through increasing high-density lipoprotein cholesterol (HDL-C) and apolipoprotein AI (Apo-AI) levels in patients with hyperlipidemia. 168][19][20][21] A single-center randomized controlled trial found that flavonoids suppressed the growth of existing atherosclerotic plaques by 1.5-fold in postmenopausal women after 12-month follow-up. 22o date, clinical trials on flavonoids for the prevention and treatment of atherosclerosis are still scarce, and the specific mechanisms of anti-atherosclerosis of flavonoids are very shallow in clinical researches.Therefore, it is necessary to synthesize preclinical studies to refine the efficacy and intervention mechanisms of flavonoids against atherosclerosis.
Herein, we conducted a systematic review and meta-analysis of data from studies to appraise the efficacy of flavonoids on animal models of atherosclerosis.The changes in atherosclerotic lesion area, serum lipid markers, and circulating inflammatory factors were included as observation parameters.Furthermore, we summarized the currently known possible mechanisms of flavonoids in the treatment of atherosclerosis.

Study selection
First, we systematically searched 8743 studies, of which 2336 duplications were removed.Then, we deleted 2138 studies by browsing the publication types.Next, based on the predefined exclusion criteria, 4125 articles were eliminated.Finally, after full-text evaluation, 31 studies were excluded, and 119 qualified articles were included in this meta-analysis.A flowchart depicting the process of selection is shown in Figure 1.

Study quality
The quality of the included studies was assessed according to the criteria of SYRCLE's risk of bias tool. 142The quality score of all the included studies ranged from 3 to 6. None of the studies specifically describe allocation concealment, blinding interventions, random outcome assessment, or blinding of outcome assessment, so they were estimated to have an unclear risk of bias.All included studies did not indicate incomplete outcome data, selective reporting, or other bias, so we estimated them to be low risk of bias.One study 33 clearly indicated grouping based on weight.Three studies 26,42,56 explicitly stated that a random number table method was used to group.The remaining studies only stated that they were randomly assigned but did not mention the specific randomization method.Baseline characteristics such as gender, age, and weight of the animals were fully described in 90.7% of the studies but were not adequately reported in 9.2% of the articles.89 publications clearly described that all experimental animals were kept in the same conditions and environment, while the other 30 studies did not provide sufficient relevant information.The results of the quality evaluation are presented in Figure 2 and Table S2.À3.15 to À1.70, P<0.00001) (Figure 6A).Heterogeneity was statistically significant (I 2 = 85%, P<0.00001) (Table S3).

Potential molecular mechanisms
The potential molecular mechanisms of various flavonoids effects on atherosclerosis in eligible studies are summarized in Table S4.

Subgroup analysis
The subgroup analysis was carried out to demonstrate the significant influencing factor of heterogeneity and determine the effect of the subclasses of flavonoids on the outcome measures.We found that the type of flavonoids may not be the potential source of heterogeneity (Table S3).

Sensitivity analysis
For atherosclerotic lesion area (longitudinal and cross-sectional plaque area), serum lipid markers (TC, TG, LDL-C, and HDL-C), and circulating inflammatory factors (TNF-a, IL-1b, IL-6, and IL-10), sensitivity analysis was performed to confirm and account for the stability of the positive results.In summary, no statistically significant change in pooled effects was detected after excluding each study separately, which suggested the results remained robust.The results of the sensitivity analysis showed that the combined effects of all outcome measures except serum IL-10 were robust (Figures 7, 8, 9, and 10).

Meta-regression analysis
Meta-regression analysis by subclass of flavonoids and species had no impact on the pooled effect size of all outcome measures (P>0.05)(Table S5).

Publication bias
As shown in Figure 11 and Table S6, results from both Begg's test and Egger's test indicated significant publication bias for all outcome measures (P<0.05).However, further analysis with trim-and-fill computation demonstrated that publication bias did not change the pooled effect sizes (Figure S1).

DISCUSSION
Flavonoids are plant secondary metabolites widely found in nature. 13They maintain lipid homeostasis by regulating lipid metabolism, which is, increasing reverse cholesterol transport or HDL-C, or protecting against HDL dysfunction. 143,144Flavonoids also play important roles in inhibiting the onset and development of inflammatory diseases by exerting their powerful anti-inflammatory properties via inhibiting regulatory enzymes or transcription factors involved in inflammation. 145Furthermore, flavonoids can regulate apoptosis through the modulation of the levels of pro-and anti-apoptosis proteins. 146Accumulating evidence demonstrates that flavonoids can scavenge free radicals and inhibit metal-ion chelators to exert antioxidant effect. 147Additionally, flavonoids can improve the functions of endothelial (ECs) and vascular smooth muscle cells (VSMCs) by inhibiting or stimulating diverse ion channels. 148On account of their multiple biological activities, flavonoids have been applied to prevent and treat cancer and cardiovascular diseases. 149Atherosclerosis is a chronic lipid-driven inflammatory disease of large and medium-sized arteries. 2,5In atherosclerosis patients, anti-inflammatory therapy may reduce thrombotic risk, 150 and maintaining optimal lipid levels can minimize the rate of progression of atherosclerotic plaques. 151herefore, we presume that flavonoids can ameliorate atherosclerosis.In this study, a comprehensive meta-analysis of existing preclinical studies on the efficacy of flavonoids in atherosclerosis was performed.The results of the included studies demonstrated that flavonoids significantly decreased serum lipid levels, plasma pro-inflammatory factor concentrations, and atherosclerotic lesion area in animal models.

Lipid-lowering effects and mechanisms of flavonoids
It is well known that hypercholesterolemia, one of the major risk factors for the occurrence and development of atherosclerosis, is one of the main therapeutic targets of atherosclerosis at present.Epidemiological data show that TG has a causal relationship with the development of atherosclerosis. 152There is substantial evidence that a high level of plasma LDL-C contributes to the progression of atherosclerosis. 153In contrast, the level of plasma HDL-C is negatively correlated with atherosclerosis. 154The reverse cholesterol transport process, an endogenous mechanism that allows cells to export cholesterol, is crucial for the maintenance of lipid homeostasis.ATP-binding cassette subfamily A member 1 (ABCA1) and G member 1 (ABCG1) are critical receptors for the initial and rate-limiting steps of reverse cholesterol transport and contribute to cholesterol efflux in foam cells. 155,156In the process, ABCA1 mediates the initial transport of cellular cholesterol to apolipoprotein A-I for forming nascent HDL particles, and ABCG1 promotes subsequent continued cholesterol efflux.Liver X receptors (LXR) regulate both ABCA1 and ABCG1, and facilitate reverse cholesterol transport. 157Studies have shown that flavonoids can promote cholesterol efflux by up-regulating the LXRa-ABCA1/ABCG1 pathway, thereby reducing foam cell formation. 38,56,64,124,136here is clear evidence that proprotein convertase subtilisin kexin type 9 (PCSK9) plays a crucial role in the maintenance of lipid homeostasis.PCSK9, a serine protease belonging to the proprotein convertase family, is mainly produced by the liver. 158LDL receptor (LDLR) is a key factor in the regulation of plasma cholesterol levels that clears circulating LDL-C via clathrin-mediated endocytosis. 159PCSK9 promotes the   degradation of LDLR by targeting this receptor to the lysosome 160 and inhibits LDLR recirculation to the cell surface. 161When the catalytic domain of PCSK9 binds to LDLR, the Cys-His-rich domain (CHRD) of PCSK9 interacts with cyclase-associated protein-1 (CAP-1) to lead the LDLR/PCSK9/CAP1 complex to lysosomal degradation through the caveolin-dependent pathway. 162In addition, PCSK9 inhibits ABCA1mediated reverse cholesterol transport through downregulation of ABCA1. 163A preclinical investigation indicated that epigallocatechin gallate up-regulated LDLR expression by suppressing PCSK9 production, resulting in lowering LDL-C levels. 164Wang et al. found that naringin promoted reverse cholesterol transport by inhibiting PCSK9 in APOE À/À mice. 108Studies have shown that quercetin accelerated cholesterol efflux by down-regulating PCSK9 to increase ABCA1 expression. 42,56ti-inflammatory effects and mechanisms of flavonoids Lipid accumulation in macrophages induces inflammation, which in turn promotes and accelerates the development of atherosclerosis. 165nflammation is involved in the initiation and development of atherosclerotic plaques. 166In the initial stage of atherosclerosis, LDL-C accumulate in the intima and activates the endothelium.Injured endothelial cells release chemokines and adhesion, attract monocytes to adhere to the endothelium, and then macrophages infiltrate into the subendothelium to form foam cells. 167Many cytokines, such as TNF-a, IL-1b, IL-6, and IL-10, are involved in this process.TNF-a, IL-1b, and IL-6 are pro-inflammatory cytokines, and the pro-inflammatory response mediated by them augments plaque growth and instability. 167,168IL-10 is an anti-inflammatory cytokine, and the major roles which IL-10 protects against atherogenesis include reduction of apoptosis, inhibition of pro-inflammatory cytokines, and regulation of lipid homeostasis. 169Tolllike receptor 4 (TLR4), a pattern recognition receptor for innate immunity, induces the production of pro-inflammatory cytokines through activating nuclear factor-kB (NF-kB). 170In addition, TLR4 negatively regulates ABCG1, which is also a key gene that mediates inflammation. 171F-kB family are key regulators of inflammation in atherosclerosis. 172Moreover, NF-kB activation in macrophages mediates foam cell biogenesis. 173Heme oxygenase-1 (HO-1), encoded by the gene HMOX1 in humans, 174 is the first and rate-limiting inducible enzyme of heme degradation 175 and exhibits anti-inflammatory properties. 176The transcription factor NF-kB can bind the HMOX1 promoter to upregulate HO-1. 177Besides, HO-1 transcription is initiated by activated peroxisome proliferator-activated receptor g (PPARg), which is translocated to further activate PPAR response elements (PPREs). 178The silent information regulator sirtuin 1 (SIRT1) plays a role in anti-inflammation by inhibiting NF-kB signaling via deacetylating the p65 subunit of NF-kB complex. 179Moreover, SIRT1 activity is promoted by AMP-activated protein kinase (AMPK) via increasing intracellular NAD+ levels. 180Interestingly, SIRT1 is a target gene of PPAR-a and is suppressed by PPARa. 181Zhao et al. suggested that astragalin retards atherosclerosis by inhibiting the inflammatory response via down-regulating the TLR4/NF-kB pathway. 136Liu et al. demonstrated that kuwanon G improved inflammation by reducing the activity of NF-kB. 64Yu et al. found that biochanin A reduced inflammation by up-regulating the PPARg/HO-1 pathway. 124Li et al. reported that luteolin prevented plaque development by decreasing macrophage inflammation via decreasing the AMPK-SIRT1 signaling. 55

Effects and mechanisms of flavonoids on reducing atherosclerotic plaque area
It is generally accepted that ECs, macrophages, and VSMCs play the predominant roles in the pathogenesis of atherosclerosis. 182When activated by stimuli related to cardiovascular risk factors, endothelial cells can secrete critical leukocyte adhesion molecules, such as vascular cell adhesion molecular-1 (VCAM-1), to promote circulating monocytes to the endothelial layer. 182Mediated by chemoattractant cytokines, macrophages migrate into the intima, and engulf the lipid, transforming into foam cells.Besides, VSMCs in the tunica media, in response to leukocyte mediators, migrate into the intima and give rise to foam cells.During the progression of atherosclerotic lesion, VSMCs produce extracellular matrix (such as interstitial collagen) that increases the thickness of the intimal layer.Furthermore, activated macrophages can increase the secretion of matrix metalloproteinases (MMPs) that degrade interstitial collagen. 183As the lesion advances, foam cells can undergo cell death, and the apoptotic cells release the lipid, which exacerbates inflammation and oxidative stress. 184A study showed that cyanidin-3-Oglucoside could attenuate endothelial cell dysfunction by inhibiting miR-204-5p/SIRT1-mediated inflammation and apoptosis. 109Chen et al. found that corylin could reduce the formation of atherosclerotic plaque by decreasing the production of VCAM-1 to inhibit monocyte adhesion via down-regulating the ROS/JNK pathway. 28Wang et al. reported that icariin could inhibit atherosclerosis progress through reducing macrophage infiltration via down-regulating the expression of CX3C chemokine receptor 1 (CX3CR1). 104Zhang et al. demonstrated that icariin contributed to plaque stabilization by facilitating collagen accumulation via inhibiting interstitial collagenase-1 (MMP-1). 130Wang et al. found that apple procyanidins significantly reduced aortic intimal-medial thickness on ultrasonography and the lipid accumulation area stained with Sudan IV in a rabbit model. 185A preclinical study demonstrated that quercetin decreased the aortic lesions by 20-70% based on ultrasound biomicroscopy analyses. 54Two observational studies suggested that pycnogenol reduced the arterial lesion progression that was evaluated using the ultrasonic arterial score based on the arterial wall morphology and the number of plaques that progressed. 54,186,187

Conclusions
In animal models, flavonoids decreased the levels of serum lipids and circulating pro-inflammatory factors, thereby reducing the atherosclerosis plaque size.In terms of molecular mechanisms, flavonoids improved lipid metabolism through various mechanisms, including the LXRa-ABCA1/ABCG1 pathway and the PCSK9-LDLR signaling, to exert anti-atherosclerosis functions.Moreover, flavonoids exhibited antiatherosclerosis properties through anti-inflammatory mechanisms like the TLR4/NF-kB pathway and the AMPK-SIRT1 signaling.Besides, flavonoids reduced the atherosclerosis plaque size by diverse mechanisms, such as the ROS/JNK pathway and the CX3CR1 signaling, leading to anti-atherosclerosis effects.In summary, flavonoids exerted anti-atherosclerosis effects by reducing the levels of serum lipids and circulating pro-inflammatory factors and the size of atherosclerotic plaque, which provides evidence for the potential cardiovascular benefits of flavonoids.

Limitations of the study
First, preclinical studies only suggest a potential for cardioprotection by flavonoids, and there is a need for clinical confirmation of these findings if they are to be considered valid.Second, considering the heterogeneity, the results must be explained circumspectly.The influence of flavonoid types and animal species on heterogeneity was basically excluded, but other possible influencing factors of heterogeneity, such as gender and age of animals and dose and duration of administration, were not fully analyzed due to insufficient information in the included literatures.In addition to the restrictive factors mentioned previously, there are still other unpredictable factors in the included studies that need to be further verified by more experiments.Third, the efficacy of different subclasses of flavonoids was not compared.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Figure 1 .
Figure 1.Flowchart of the search process

Figure 2 .
Figure 2. Risk of bias and quality evaluation

Figure 3 .
Figure 3. Forest plots of flavonoids effect on atherosclerotic lesion area (A) longitudinal plaque area; (B) cross-sectional plaque area.

Figure 7 .
Figure 7. Sensitivity analysis of flavonoids effect on atherosclerotic lesion area (A) longitudinal plaque area; (B) cross-sectional plaque area.

Figure 8 .
Figure 8. Sensitivity analysis of flavonoids effect on serum lipid markers (A) TC; (B) TG.

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS d METHOD DETAILS B Search Strategy B Inclusion and exclusion criteria B Data extraction B Quality assessment d QUANTIFICATION AND STATISTICAL ANALYSIS d ADDITIONAL RESOURCES